Transcriptional activation involves an interplay of sequence- specific transcription factors bound to the promoter DNA and the components of the basal transcription machinery. Defining the precise mechanism by which site-specific transcription factors activate transcription has been the subject of intense investigation. It has been proposed that transcriptional activators may target multiple steps during the assembly of a preinitiation complex. One such target appears to be TFIID, a multi-subunit complex consisting of the TATA box-binding protein (TBP) and multiple tightly associated factors (TAFs), which serve as coactivators for mediating transcriptional activation. The general objective of the proposed research is to identify and characterize the functional relationship between TAFs and activators. The long-term goal is to reconstitute transcription with recombinant TFIID subunits and activators so that the molecular events leading to transcriptional activation may be defined more precisely. The first specific aim involves examination of the functional properties of the recently cloned human TAF130. Since TAFs function as coactivators, it is predicted that they directly contact the activators. Preliminary studies suggest that hTAF130 binds to the human transcription factor Sp1 in vitro. The domains of hTAF130 and Sp1 required for binding will be characterized by affinity chromatographic methods and genetic screens in yeast. Other activators will be tested for binding to TAF130 and their interactions characterized. The significance of these TAF-activator interactions will be examined in the context of transcription. Towards this end, a partial TFIID complex reconstituted in vitro with purified recombinant TAF proteins and TBP will be tested in transcription assays in the presence of the activators that bind to TAF130. The second specific aim is to devise a system in vivo using vaccinia viral vectors for efficient assembly of the TFIID complex. For analytical purposes, subunits of TFIID will be transiently expressed in HeLa cells using the infection/transfection protocol and complex assembly will be monitored by pulse-labeling and immunoprecipitation. A potential advantage of this approach is the efficiency and ease with which the mutant proteins may be analyzed for function. For the large scale production of the recombinant proteins, HeLa cells will be coinfected with the recombinant viruses expressing each subunit of TFIID and the partial complexes will be purified for further biochemical analyses including in vitro transcription and DNA binding assays. The third specific aim is to identify the target of the cloned human TAF95 protein using the GAL4 two hybrid system in yeast. A human cDNA expression library will be used in the screen to identify the potential interacting target(s) of hTAF95. hTAF95 contains several copies of the WD40 repeat, a motif implicated in protein-protein interactions. Candidate clones will be tested for interaction with hTAF95 in vitro and their function examined in transcription assays. The proposed research may shed some light into the general mechanism of transcriptional activation. These findings are relevant to defining the molecular events occurring during normal cell growth, differentiation and development, as well as in cancer and other human diseases.